JP7469812B2 - Compounds with antimicrobial properties - Google Patents

Description

This application claims priority to U.S. Provisional Patent Application No. 62/700,746, filed July 19, 2018, and U.S. Provisional Patent Application No. 62/789,313, filed January 7, 2019, which are incorporated herein by reference.

This disclosure relates to novel antimicrobial compounds and analogs thereof, pharmaceutical compositions containing said compounds, and therapeutic uses of said compounds and pharmaceutical compositions. This invention was made with Government support under Grant No. P01-AI118687 awarded by the National Institutes of Health. The Government has certain rights in this invention.

Certain Gram-negative bacteria can cause serious complications and infections such as pneumonia, urinary tract infections, wound infections, ear infections, eye infections, intra-abdominal infections, oral bacterial overgrowth and sepsis in susceptible individuals. Treatment of serious bacterial infections in clinical practice can be complicated by antibiotic resistance. In recent years, there has been an increase in infections caused by Gram-negative bacteria that are resistant to multiple classes of antibiotics, including broad-spectrum antibiotics such as aminoglycosides, cephalosporins and even carbapenems. This alarming trend highlights the need to identify new antibiotics that are effective against Gram-negative bacteria, especially multidrug-resistant Gram-negative bacteria.

Polymyxins are a class of antibiotics produced by the Gram-positive bacterium Bacillus polymyxa. First identified in the late 1940s, polymyxins, particularly polymyxin B and polymyxin E (colistin), were once used in the treatment of Gram-negative infections. However, these antibiotics exhibited side effects, including nephrotoxicity. As a result, their use in therapy was limited to treatments of last resort.

The present invention relates to novel compounds and analogs thereof that exhibit superior antibacterial activity, particularly against gram-negative pathogens. Pharmaceutical compositions containing the novel compounds and analogs thereof are useful for treating or preventing a variety of infectious diseases, including skin and skin structure infections, respiratory infections, sepsis, bacteremia, and inflammatory bowel disease (IBD). Embodiments of the present invention also relate to the treatment or prevention of bacterial infections.

In one aspect, the present invention relates to a compound represented by the following formula (II):

and/or its pharma- ceutically acceptable salts, stereoisomers (including enantiomers), tautomers, or hydrates, as well as analogs of formula (II), including novel compounds represented by formula (II) (hereinafter collectively referred to as "formula (II) compounds"). The present invention also includes pharmaceutical compositions comprising or consisting essentially of formula (II) compounds, uses of formula (II) compounds, and methods of treating or preventing bacterial infections using one or more formula (II) compounds.

In another aspect, the present invention provides a compound of formula (I):

(Wherein:
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, hydroxyl, hydroxyalkyl, halogen, -CN, -O-alkyl, -C(O)-alkyl, -C(O)O-alkyl, -C(O)OH, -C(O)NH ₂ , -C(O)NH-alkyl, -NH ₂ , -NO ₂ , -CF ₃ , -NH-alkyl, -N-(alkyl) ₂ , -NHC(O)-alkyl, and aryl, each of which may be substituted;
R ₇ and R ₈ are each independently selected from the group consisting of hydrogen, straight or branched C1-C5 alkyl, straight or branched C2-C6 alkenyl, straight or branched C2-C6 alkynyl; each of said alkyl, alkenyl, and alkynyl may be optionally substituted;
R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , R ₁₅ , R ₁₆ , R ₁₇ , R ₁₈ , R ₁₉ and R ₂₀ are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, haloalkyl, hydroxyl, hydroxyalkyl, halogen, -CN, -O-alkyl, -C(O)-alkyl, -C(O)O-alkyl, -C(O)OH, -C(O)NH ₂ , -C(O)NH-alkyl, -NH ₂ , -NO ₂ , -CF ₃ , -NH-alkyl, -NHC(O)-alkyl; wherein said alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, haloalkyl are each optionally substituted;
A is a bond or -(CH ₂ ) _n -, where n is an integer from 0 to 10;
X is -C(O)-, _-CH2- , -C(OH)-, -C(O)O-alkyl, -C((O)alkyl)-.
and/or its pharma- ceutically acceptable salts, stereoisomers (including enantiomers), tautomers, or hydrates, as well as analogs thereof, including novel compounds represented by formula (I) (hereinafter collectively referred to as "formula (I) compounds"). The present invention also includes pharmaceutical compositions comprising or consisting essentially of formula (I) compounds, uses of formula (I) compounds, and methods of treating or preventing bacterial infections using one or more formula (I) compounds.

The compounds of formula (I), their prodrugs, pharma- ceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, and racemates exhibit advantageous antibacterial activity against a variety of Gram-negative bacteria and are useful in the treatment or prevention of a variety of infectious diseases induced by a variety of bacteria in humans and other animals.

The compound of formula (I) has the following formula (Ia):

(Wherein:
R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are as defined above.
and/or its pharma- ceutically acceptable salts, stereoisomers (including enantiomers), tautomers, or hydrates, and analogs of formula (Ia) (hereinafter collectively referred to as "formula (Ia) compounds"). The present invention also includes pharmaceutical compositions comprising or consisting essentially of formula (Ia) compounds, uses of formula (Ia) compounds, and methods of treating or preventing bacterial infections with one or more formula (Ia) compounds.

Another aspect of the invention relates to pharmaceutical compositions for the treatment of infections in animals caused by Gram-negative bacteria. In various embodiments, the pharmaceutical compositions comprise a therapeutically effective amount of one or more compounds of formula (I), (Ia) or (II), or a pharma- ceutically acceptable salt thereof, and one or more additional compounds selected from the group consisting of pharma- ceutically acceptable carriers, surfactants, solid diluents, and liquid diluents. The compounds of formula (I), (Ia) or (II) may optionally be present in the form of a pharma- ceutically acceptable solvate, such as a hydrate.

Generally, as used herein, the term "substantially" means ±10%, and in some cases ±5%. Additionally, throughout this specification, reference to "one example," "an example," "one embodiment," or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the example is included in at least one example of the technology. Thus, the phrases "in one example," "in an example," "one embodiment," or "an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same example. Furthermore, particular features, structures, routines, steps, or characteristics may be combined in any suitable manner in one or more examples of the technology. The headings provided herein are for convenience only and are not intended to limit or construe the scope or meaning of the claimed technology.

Illustrated is a reverse phase high performance liquid chromatography (RP-HPLC) chromatogram monitoring the compound of formula (II) at 254 nm with a retention time of 12.25 minutes. 1 illustrates the chemical structure of the compound according to formula (II). 1 is a graph showing the results of in vitro activity of the compound of formula (II) against E. coli. The compound of formula (II) exhibited bactericidal activity. E. coli was grown to early exponential phase and treated with 2x the MIC of the compound of formula (II). After 20 hours of incubation, E. coli cultures were plated for viable cell counts. Schematic representation of the Darobactin A biosynthetic gene cluster and sequence comparison. FIG. 5A is a graph showing the results of the in vivo efficacy of Darobactin A in an E. coli sepsis model. The graph shows the survival rate of mice with E. coli sepsis. Darobactin A was able to cure both ATCC25922 or polymyxin-resistant clinical isolate AR350 E. coli sepsis mice at 2.5 mg/kg (ip) (n=3 per treatment group). Treatment was 1 hour after infection. At 1 mg/kg (ip), Darobactin A extended survival time and significantly reduced CFU load in the blood at 5 hours. Gentamicin was used as a positive control and given at 20 mg/kg. Figure 5B is a graph showing the results of colony forming units/milliliter (CFU/mL) in blood of mice with E. coli sepsis. Darobactin A at 2.5 mg/kg (ip) was able to cure mice with both ATCC25922 or polymyxin-resistant clinical isolate AR350 E. coli sepsis (n=3 per treatment group). Treatment was 1 hour after infection. At 1 mg/kg (ip) Darobactin A extended survival time and significantly reduced CFU burden in blood at 5 hours. Gentamicin was used as a positive control and given at 20 mg/kg. Figure 5C is a graph showing the results of the in vivo efficacy of Darobactin A in a P. aeruginosa sepsis model. Darobactin A was tested at 50 mg/kg against P. aeruginosa P7-PA01 (a polymyxin-resistant laboratory isolate) sepsis and was able to completely cure the mice (n=3 per group). Gentamicin was used as a positive control and was given at 20 mg/kg. Figure 5D is a graph showing the results of the in vivo efficacy of Darobactin A in an E. coli neutropenic thigh infection model. In the thigh model, 50 mg/kg (ip) Darobactin A at 2 hours post-infection reduced CFU burden in the thigh 10-fold compared to untreated controls at 24 hours, and 25 mg/kg (ip) given three times at 2, 8, and 14 hours reduced CFU burden 100-fold, including below the limit of detection in two mice. Gentamicin was used as a positive control and given at 20 mg/kg. 1 is a graph showing the results of in vivo efficacy of the compound of formula (II) in a mouse gastrointestinal infection with Salmonella Typhimurium.

As used herein, unless otherwise specified, the term "compounds of the invention" collectively refers to the compounds of formula (I), (Ia) and (II) and pharma- ceutically acceptable salts thereof and the specific compounds depicted herein. The compounds of the invention are identified herein by chemical structure and/or chemical name. When a compound is represented by a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the identity of the compound. The compounds of the invention may contain one or more chiral centers and/or double bonds and therefore may exist as stereoisomers, such as double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. In accordance with the present invention, the chemical structures depicted herein, and thus the compounds of the invention, encompass all of the enantiomers and stereoisomers of the corresponding compounds, i.e., both stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be separated into their component enantiomers or stereoisomers by well-known methods such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods. The compounds of the present invention are effective against important gram-negative pathogens. These compounds have excellent activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella Typhimurium and Shigella sonnei. Furthermore, the compounds of the present invention have no activity against gram-positive pathogens and are inactive against gram-negative intestinal flora, Bacteroides. As will be explained below, this selectivity is pharmacologically beneficial. Based on their unusual structure, which contains a C-C bond between tryptophan and lysine, the compounds of the present invention belong to a new class of antibacterial agents. In fact, the last new class of compounds acting against gram-negative bacteria was discovered 50 years ago.

Selective activity against Gram-negative pathogens is extremely rare; in fact, there is only one known compound with such properties - polymyxin, which acts by binding bacterial lipopolysaccharide (LPS). The compounds herein do not bind LPS and, importantly, are active against polymyxin-resistant mutants. Polymyxins are antibacterial drugs of last resort against multidrug-resistant (MDR) Gram-negative bacteria.

The compounds of the invention have very low tolerance and no detectable cytotoxicity. They may be used to treat local and systemic infections with gram-negative pathogens, such as skin and skin structure infections, respiratory infections, sepsis and bacteremia.

Another important application of the compounds of the invention results from their selective activity against the pathogenic commensal bacteria of the large intestine. In patients with inflammatory bowel disease (IBD), the Enterobacteriaceae flora, specifically the E. coli flora, fuels the inflammatory cycle. Broad-spectrum antibacterial drugs have limited efficacy because they harm the commensal flora. The compounds of the invention are inactive against Gram-positive bacteria and against Bacteroides, the major Gram-negative co-producers of the gastrointestinal tract. The compounds of the invention may be used to eliminate the gut flora of IBD patients. The prevalence of Enterobacteriaceae is also a common feature of a dysbiotic gut and is treated with one or more of the compounds of the invention.

As used herein, unless otherwise specified, the term "alkyl" means a substituted or unsubstituted, saturated, straight or branched hydrocarbon chain radical. Examples of alkyl groups include, but are not limited to, C 1-15 straight, branched or cyclic alkyls such as methyl, ethyl, propyl, isopropyl, cyclopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl- _1- butyl, butyl, isobutyl, sec-butyl, t-butyl, cyclobutyl, pentyl, isopentyl, neopentyl, hexyl, and cyclohexyl, as well as long chain alkyl groups such as heptyl, octyl, nonyl and decyl. An alkyl can be unsubstituted or substituted with one or two suitable substituents.

As used herein, unless otherwise specified, the term "alkoxy" or "alkyloxy" means -O-alkyl, where alkyl is as defined herein. Alkoxy can be unsubstituted or substituted with one or two suitable substituents. Preferably, the alkyl chain of the alkyloxy is 1-5 carbon atoms in length and is referred to herein, for example, as "C _1-5 alkoxy." In one embodiment, the alkyl chain of the alkyloxy is 1-10 carbon atoms in length and is referred to herein, for example, as "C _1-10 alkoxy."

As used herein, unless otherwise specified, the term "alkene" or "alkenyl group" means a monovalent straight, branched, or cyclic hydrocarbon chain having one or more double bonds therein. The double bond of an alkene can be unconjugated or conjugated to another unsaturated group. The alkene can be unsubstituted or substituted with one or two suitable substituents. Suitable alkenes include, but are not limited to, C2-8 alkenyl groups such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl- ₂ -butenyl, 4-(2-methyl-3-butene)pentenyl, and the like. The alkene can be unsubstituted or substituted with one or two suitable substituents.

As used herein, unless otherwise specified, the term "alkynyl" means an unsaturated straight or branched chain hydrocarbon having at least one carbon-carbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl.

As used herein, unless otherwise specified, the term "aryl" or "aromatic ring" refers to a monocyclic or polycyclic conjugated ring structure well known in the art. Examples of suitable aryl groups or aromatic rings include, but are not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl. The aryl group may be unsubstituted or substituted with one or two suitable substituents. In one embodiment, the aryl group is a monocyclic ring, and the ring contains 6 carbon atoms, referred to herein as " _C6 aryl".

"Substituted aryl" includes halo, alkyl, haloalkyl (e.g., trifluoromethyl), alkoxy, haloalkoxy (e.g., difluoromethoxy), alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, aryloxy, aryloxyalkyl, arylalkoxy, alkoxycarbonyl, alkylcarbonyl, arylcarbonyl, arylalkenyl, aminocarbonylaryl, arylthio, arylsulfinyl, arylazo, heteroarylalkyl, heteroarylalkenyl, heteroaryloxy, hydroxy, nitro, cyano, amino, amino is 1 or 2 substituents (optionally substituted alkyl, aryl or and/or one or more functional groups such as substituted amino, thiol, alkylthio, arylthio, heteroarylthio, arylthioalkyl, alkoxyarylthio, alkylaminocarbonyl, arylaminocarbonyl, aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, alkylcarbonylamino, arylcarbonylamino, arylsulfinyl, arylsulfinylalkyl, arylsulfonylamino, or arylsulfoneaminocarbonyl, which may be substituted with any of the alkyl substituents described herein.

As used herein, alone or as part of another group, the term "heteroaryl" refers to 5-7 membered aromatic rings containing 1, 2, 3 or 4 heteroatoms such as nitrogen, oxygen or sulfur, as well as such rings fused with an aryl, cycloalkyl, heteroaryl or heterocycloalkyl ring (e.g., benzothiophenyl, indolyl), including possible N-oxides. "Substituted heteroaryl" includes heteroaryl groups that may be substituted with 1 to 4 substituents, such as those included above in the definitions of "substituted alkyl" and "substituted cycloalkyl". Substituted heteroaryl also includes fused heteroaryl groups, including, for example, quinoline, isoquinoline, indole, isoindole, carbazole, acridine, benzimidazole, benzofuran, isobenzofuran, benzothiophene, phenanthroline, purine, and the like.

Additionally, as used herein, the terms "heterocyclo", "heterocycle", or "heterocyclic ring" refer to an unsubstituted or substituted stable 5- to 7-membered monocyclic ring structure, which may be saturated or unsaturated, and which consists of carbon atoms and from 1 to 4 heteroatoms selected from N, O, or S, wherein the nitrogen and sulfur heteroatoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. The heterocyclic ring may be attached at any heteroatom or carbon atom that results in the creation of a stable structure. Examples of such heterocyclic groups include, but are not limited to, piperidinyl, piperazinyl, oxopiperazinyl, oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, pyrrolyl, pyrrolidinyl, furanyl, thienyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, thiadiazolyl, tetrahydropyranyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl.

As used herein, the term "optionally substituted" means that, for example, a chemical moiety representing an alkyl, aryl, heteroaryl may be unsubstituted or may be substituted with one or more groups including, but not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl, aryl, heterocyclic, heteroaryl, hydroxyl, amino, alkoxy, halogen, carboxy, carbalkoxy, carboxamido, monoalkylaminosulfinyl, dialkylaminosulfinyl, monoalkylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino, hydroxysulfonyloxy, alkoxysulfonyloxy, alkylsulfonyloxy, hydroxysulfonyl, alkoxysulfonyl, alkylsulfonylalkyl, monoalkylaminosulfonylalkyl, dialkylaminosulfonylalkyl, monoalkylaminosulfinylalkyl, dialkylaminosulfinylalkyl, and the like. The chemical moiety of formula I may be an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl, aryl, heterocyclic, or heteroaryl. For example, an optionally substituted alkyl may include both propyl and 2-chloropropyl. In addition, "optionally substituted" also includes embodiments where the named substituent or substituents have multiple substituents rather than simply a single substituent. For example, optionally substituted aryl may include both phenyl and 3-methyl-5-ethyl-6-chlorophenyl.

The term "cycloalkyl" includes saturated or partially unsaturated (containing one or more double bonds) cyclic hydrocarbon groups containing one to three rings, including monocyclic alkyl, bicyclic alkyl, and tricyclic alkyl, and containing a total of 3 to 20 carbons forming the ring, or about 3 to 10 carbons forming the ring and optionally fused with one or two aromatic rings as described for aryl.

"Substituted cycloalkyl" includes cycloalkyl groups that may be substituted with one or more substituents such as halogen, alkyl, substituted alkyl, alkoxy, hydroxy, aryl, substituted aryl, aryloxy, cycloalkyl, alkylamido, alkanoylamino, oxo, acyl, arylcarbonylamino, amino, nitro, cyano, thiol, and/or alkylthio, and/or any of the substituents included in the definition of "substituted alkyl."

The term "cycloalkenyl" includes non-aromatic monocyclic or bicyclic carbocyclic rings containing at least one double bond. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

As used herein, unless otherwise specified, the term "aryloxy" means an -O-aryl group, where aryl is as defined herein. An aryloxy group can be unsubstituted or substituted with one or two suitable substituents. Preferably, the aryloxy group is a monocyclic ring, the ring containing 6 carbon atoms, referred to herein as " _C6 aryloxy."

As used herein, unless specifically stated otherwise, the term "ether" means a group of formula alkyl-O-alkyl, alkyl-O-alkynyl, alkyl-O-aryl, alkenyl-O-alkenyl, alkenyl-O-alkynyl, alkenyl-O-aryl, alkynyl-O-alkynyl, alkynyl-O-aryl, aryl-O-aryl, where "alkyl", "alkenyl", "alkynyl" and "aryl" are defined herein.

As used herein, unless otherwise specified, the term "carboxy" refers to a radical of the formula: -COOH.

As used herein, unless otherwise specified, the term "halogen" means fluorine, chlorine, bromine, or iodine. Similarly, the meaning of the terms "halo" and "Hal" includes fluoro, chloro, bromo, and iodo.

As used herein, unless specifically stated otherwise, the terms "substituted" and "suitable substituent" mean groups that do not abolish the synthetic or pharmaceutical utility of the compounds of the invention or intermediates useful in making them. Examples of substituents or suitable substituents include C _1-10 alkyl; C _1-10 alkenyl; C _1-10 alkynyl; C ₆ aryl; C _3-5 heteroaryl; C _3-7 cycloalkyl; C _{1-10 alkoxy; C 6 aryloxy} ; -CN; -OH; SH; oxo; halo; -NO ₂ ; -CO ₂ H; -NH ₂ ; -NHOH; -NH(C _1-10 alkyl); -N(C _1-10 alkyl) ₂ ; -NH(C ₆ aryl); -NHO(C _1-10 alkyl); -N(OC _1-10 alkyl) ₂ ; -NH(OC ₆ aryl); -S(C _1-10 alkyl); -S(C ₆ aryl); (=O); -N(C ₆ aryl) ₂ ; -CHO; -C(O)(C Examples of such aryl groups include, but _{are not limited to, -C(O)(C 1-10 alkyl} ); -C(O)O(C _1-10 alkyl); and -C(O)O(C ₆ aryl), -C(S)(C _1-10 alkyl); -C(S)(C ₆ aryl); -SO ₂ (C _1-10 alkyl); -SO ₂ (C ₆ aryl), -SO(C _1-10 alkyl); -SO(C ₆ aryl), and -SO ₃ H, -C(S)O(C _1-10 alkyl); -C(S)OC ₆ aryl. In certain illustrative embodiments, the substituents can be one or more suitable groups such as, but not limited to, -F, -Cl, -Br, -I, -OH, azido, -SH, alkyl, aryl, heteroalkyl, alkyloxy, alkylthiol, amino, hydroxylamino, N-alkylamino, -N,N-dialkylamino, -N,N-dimethylamino, acyl, alkyloxycarbonyl, sulfonyl, urea, -NO ₂ , and triazolyl. One of skill in the art can readily select appropriate substituents based on the stability and pharmaceutical and synthetic activity of the compounds of the invention.

As used herein, the phrase "pharmaceutical acceptable salts" includes, but is not limited to, salts of acidic or basic groups that may be present in compounds used in the present compositions, including the compounds of the present invention. Compounds included in the present compositions that are basic in nature can form a wide range of salts with various inorganic and organic acids. Acids that may be used to prepare pharmaceutical acceptable acid addition salts of such basic compounds include those that form non-toxic acid addition salts, i.e., but are not limited to, sulfuric acid, citric acid, maleic acid, acetic acid, oxalic acid, hydrochloride, hydrobromide, hydroiodide, nitrate, oxalate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, Salts containing pharma- ceutically acceptable anions include ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylenebis(2-hydroxy-3-naphthoic acid) salts). Compounds contained in the present compositions that contain an amino moiety may form pharma-ceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds contained in the present compositions that are acidic in nature are capable of forming base salts with various pharma-ceutically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts.

As used herein, the term "pharmaceutical acceptable prodrug" refers to a derivative of a compound that can undergo hydrolysis, oxidation, or other reactions under biological conditions in vitro or in vivo to obtain a compound. Examples of prodrugs include, but are not limited to, compounds that contain a biohydrolyzable moiety, such as biohydrolyzable amide, biohydrolyzable ester, biohydrolyzable carbamate, biohydrolyzable carbonate, biohydrolyzable ureide, and biohydrolyzable phosphate analogs. Other examples of prodrugs include, but are not limited to, oligonucleotides, peptides, lipids, aliphatic and aromatic groups, or compounds that contain NO, _NO2 , ONO, and _ONO2 moieties. Prodrugs can usually be prepared using well-known methods.

In one aspect, provided herein is a method for treating, ameliorating, or preventing a bacterial infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of at least one compound of Formula (I), Formula (Ia), or Formula (II), or at least one of the specific compounds described herein. Administration of the compound may be local, such as subcutaneous, transdermal, rectal, intravaginal, intranasal, intrabronchial, intraocular, or intraaural. Alternatively, administration may be systemic, such as oral administration. In yet another alternative, administration may be parenteral, intravenous, intramuscular, or intraperitoneal.

As used herein, the term "administration" can also include administering a combination of compounds. Thus, administration can be in the form of administering a compound or combination of compounds to an organism so that the circulatory system of the organism delivers the compound or combination of compounds to a target area, including, but not limited to, a cell or cells, synaptic junctions, and circulation. Administration can also mean directly contacting a compound or combination of compounds with an organ, tissue, region, area, cell, or group of cells, such as, but not limited to, directly injecting a combination of compounds.

In selected embodiments, a combination of compounds may be administered, and thus the individual compounds may be said to be co-administered with each other. As used herein, "co-administer" means administering each of at least two compounds during a time frame in which their respective periods of biological activity or effect overlap. Thus, the term co-administer includes sequential as well as coextensive administration of the individual compounds, at least one of which is a compound of the present invention. Thus, "administering" a combination of compounds according to some of the methods of the present invention includes sequential as well as coextensive administration of the individual compounds of the present invention. Similarly, the phrase "combination of compounds" means co-administering the individual compounds, and the phrase "combination of compounds" does not mean that the compounds must necessarily be administered contemporaneously or coextensively. In addition, the route of administration of the individual compounds need not be the same.

As used herein, the terms "treat" and "treatment" refer to slowing the progression of or recovering from a disease or infection. Treating a disease includes treating and/or reducing the symptoms of a disease or infection. The term "preventing" refers to slowing the disease or slowing the onset of a disease, infection, or a symptom thereof. Preventing a disease or infection can include stopping the onset of a disease, infection, or a symptom thereof.

As used herein, the term "subject" can be an animal, vertebrate, mammal, rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, simian (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human.

As used herein, the term "dosage unit" refers to a physically discrete unit, such as a capsule or tablet, suitable as a unitary administration for a subject. Each unit contains a predetermined amount of a compound of formula (I), (Ia) or (II) that has been discovered or believed to provide the desired pharmacokinetic profile that obtains the desired therapeutic effect. A dosage unit consists of a compound of formula (I), (Ia) or (II) together with at least one pharma- ceutically acceptable carrier, salt, excipient, or combination thereof. The term "dose" or "dosage amount" refers to the amount of active ingredient taken or administered to an individual at one time.

The term "therapeutically effective amount" refers to an amount sufficient to obtain a desired biological effect in a subject. Thus, a therapeutically effective amount of a compound may be an amount that, when administered to a subject suffering from or susceptible to a disease or infection, is sufficient to treat or prevent the disease or infection, and/or delay the onset or progression of the disease or infection, and/or alleviate one or more symptoms of the disease or infection. As used herein, "pharmaceutical acceptable carrier" or "pharmaceutical acceptable excipient" refers to non-API (API stands for active pharmaceutical ingredient) materials such as disintegrants, binders, and lubricants used in pharmaceutical formulations. These are generally safe for administration to humans.

The term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or carrier with which the compound of the present invention is administered. Such pharmaceutical vehicles can be liquids such as water, oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, ware, and the like. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants may be used. In one embodiment, when administered to a patient, the combination of the compound of the present invention and the pharmaceutical acceptable vehicle is sterile. When the combination of the compound of the present invention is administered intravenously, water and/or oil is one vehicle. Saline and aqueous dextrose and glycerol solutions may be used as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. If desired, the present combination of compounds can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

Generally, each of the individual compounds of the present invention may be administered by any convenient route, for example, orally, by infusion or bolus injection, or through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with another bioactive agent. Administration may be systemic or local. A variety of delivery systems are known, such as encapsulation in liposomes, microparticles, microcapsules, capsules, and the like, and may be used to administer at least one of the compounds of the present invention. Methods of administration of the individual compounds include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, pulmonary, or topical, particularly to the ear, nose, eye, or skin. The preferred method of administration is at the discretion of the practitioner and will depend, in part, on the site of the medical condition.

In certain embodiments, it may be desirable to administer one or more compounds of the combination locally to the area requiring treatment. For example, but not by way of limitation, this may be accomplished by localized injection during surgery, by topical application, e.g., with a wound dressing after surgery, by injection, using a catheter, using a suppository, or by an implant comprised of a porous, non-porous, or gelatinous material, including a membrane such as a sialastic membrane, or a fiber.

Pulmonary administration can also be used, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent, or by perfusion in a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, the compounds of the invention can be formulated as a suppository with traditional binders and vehicles such as triglycerides.

In another embodiment, the compounds of the invention can be delivered in vehicles, particularly liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327).

In yet another embodiment, at least one of the compounds used in the methods of the present invention can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507 Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, a polymeric material can be used (Medical Applications of Controlled Release,
Langer and Wise (eds.), CRC Press. , Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. See Levy et al., 23:61; , 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; see also Howard et al., 1989, J. Neurosurg. 71:105. In yet another embodiment, the controlled release system can be placed near an organ, e.g., the liver, thus requiring only a small systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science 249:1527-1533 may be used.

Each of the individual compounds to be administered can take the form of a solution, suspension, emulsion, tablet, pill, pellet, capsule, capsule containing a solution, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, or other dosage form suitable for use. In one embodiment, the pharma- ceutically acceptable vehicle is a capsule (see, e.g., U.S. Pat. No. 5,698,155). An example of a suitable pharmaceutical vehicle is described in Remington's Science,
and Practice of Pharmacy (21st ed., Hendrickson, R., et al., Eds., Lippincott Williams & Wilkins, Baltimore, MD (2006), incorporated herein by reference).

Typically, when the individual compounds of the invention are administered intravenously, they are in a sterile isotonic aqueous buffer solution. If necessary, the individual compounds of the invention may also contain a stabilizer. If necessary, the individual compounds of the invention for intravenous administration may contain a local anesthetic, such as lidocaine, to ease pain at the site of the injection.

In one embodiment, the individual compounds are supplied either together or separately in unit dosage form. In any event, the compounds may be supplied, for example, as a lyophilized powder or water-free concentrate in a sealed container such as an ampule indicating the quantity of active drug. If the compound or combination of compounds of the invention is to be administered by infusion, it can be dispensed, for example, using an infusion bottle containing sterile pharmaceutical grade water or saline. If the compound or combination of compounds of the invention is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more optional agents, e.g., sweeteners such as fructose, aspartame, or saccharin; flavorings such as peppermint, oil of wintergreen, or cherry; coloring agents; and preservatives to provide a pharma- ceutically palatable formulation. Rapid release formulations for oral use include tablets or capsules containing the active ingredient in a mixture with non-toxic pharma-ceutically acceptable excipients. These excipients may be, for example, inert diluents or binders (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, alginates, or alginic acid); binders (e.g., sucrose, glucose, mannitol, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricants, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc). Other pharma- ceutical acceptable excipients may be, for example, colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like, as may be found in The Handbook of Pharmaceutical Excipients, third edition, edited by Arthur H. Kibbe, American Pharmaceutical Association Washington DC.

Additionally, in tablet or pill form, the composition may be coated to delay disintegration and absorption in the gastrointestinal tract, providing sustained activity over an extended period of time. Selectively permeable membranes around the osmotically active driving compound are also suitable for orally administered compounds of the invention. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the drug or drug composition through an opening. These delivery platforms can provide an almost zero order delivery profile as opposed to the spiked profiles of immediate release formulations. Time-delay materials such as glycerol monostearate or glycerol stearate may be used. Oral compositions may include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and others. Such vehicles are preferably of pharmaceutical grade.

For oral delivery, the active compound can be incorporated into a formulation containing a pharma- ceutically acceptable carrier, such as a binder (e.g., gelatin, cellulose, tragacanth), an excipient (e.g., starch, lactose), a disintegrant (e.g., alginate, Primogel, and corn starch), and a sweetener or flavoring agent (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint). The formulation can be orally delivered in the form of a sealed gelatin capsule or a compressed tablet. The capsules and tablets can also be coated with various coatings known in the art to modify the aroma, taste, color, and shape of the capsules and tablets. The carrier can be solid or liquid, or both, and can be formulated with at least one compound described herein as an active compound, which can contain about 0.05% to about 95% by weight of at least one active compound. Suitable oral formulations can be in the form of a suspension, syrup, chewing gum, wafer, elixir, and the like.

Conventional agents for modifying the aroma, taste, color, and shape of a particular form may be included, if desired. In addition, the active compound may be dissolved in an acceptable lipophilic vegetable oil vehicle, such as olive oil, corn oil, and safflower oil, for convenient administration via enteral feeding tube in patients unable to swallow.

The active compounds can also be administered parenterally in the form of a solution or suspension, or in a lyophilized form that can be converted into a solution or suspension form before use. In such formulations, diluents or pharma- ceutically acceptable carriers, such as sterile water and saline buffer, can be used. Other conventional solvents, pH buffers, stabilizers, antibacterial agents, surfactants, and antioxidants can all be included. For example, useful ingredients include sodium chloride, acetate, citrate or phosphate buffers, glycerin, dextrose, fixed oils, methylparaben, polyethylene glycol, propylene glycol, sodium bisulfate, benzyl alcohol, ascorbic acid, and the like. Parenteral formulations can be stored in any conventional container, such as vials and ampoules.

Routes of topical administration include nasal, oral, mucosal, rectal, or vaginal application. For topical administration, the active compound can be formulated into lotions, creams, ointments, powders, pastes, suspensions, drops, and aerosols. Thus, one or more thickening agents, humectants, and stabilizers can be included in the formulation. Examples of such agents include, but are not limited to, polyethylene glycol, sorbitol, xanthan gum, petrolatum, beeswax, or mineral oil, lanolin, squalene, and the like. A particular form of topical administration is delivery by transdermal patch. Methods for formulating transdermal patches are disclosed, for example, in Brown, et al. (1988) Ann. Rev. Med. 39:221-229, which is incorporated herein by reference. Carriers and excipients that may be used include petrolatum, lanolin, polyethylene glycol, alcohols, and combinations of two or more thereof. The active compound is generally present at a concentration of about 0.1% to about 80% (w/w) of the composition, for example, about 0.2% to 50%.

Subcutaneous implantation for sustained release of active compounds may also be a suitable route of administration. This requires a surgical procedure to implant the active compound in a suitable formulation into the subcutaneous space, e.g., beneath the anterior abdominal wall. See, e.g., Wilson et al. (1984) J. Clin. Psych. 45:242-247. Hydrogels may be used as carriers for sustained release of active compounds. Hydrogels are generally known in the art. Hydrogels are typically prepared by crosslinking high molecular weight biocompatible polymers into a network that swells in water to produce a gel-like material. Preferably, the hydrogel is biodegradable or bioabsorbable. For purposes of the present invention, hydrogels prepared from polyethylene glycol, collagen, or glycolic acid/L-lactic acid copolymers may be useful. See, e.g., Phillips et al. (1984) J. Pharmaceut. Sci. , 73:1718-1720.

The amount of each individual compound to be administered will depend on the nature or severity of the condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may be used, as appropriate, to help determine optimal dosage ranges for each of the components of the combination. The exact dose of each component to be used will also depend on the route of administration and the severity of the disease or disorder, and the practitioner can determine these doses based on each patient's circumstances. In general, however, suitable dosage ranges for oral administration of each of the compounds of formula (I), formula (Ia), and formula (II) are typically about 0.001 mg to 1000 mg of the compound of the invention per kilogram of body weight. In certain embodiments of the invention, the oral dose of each component is 0.01 mg to 100 mg per kilogram of body weight, more particularly 0.1 mg to 50 mg per kilogram of body weight, more particularly 0.5 mg to 20 mg per kilogram of body weight, and even more particularly 1 mg to 10 mg per kilogram of body weight. In one embodiment, the oral dosage of each of the formula (I), formula (Ia), and formula (II) compounds is at least about 1, 5, 10, 25, 50, 100, 200, 300, 400, or 500 mg/day, up to 600, 700, 800, 900, or 1000 mg/day for 3 to 15 days. Each of the formula (I), formula (Ia), and formula (II) compounds may be given daily (e.g., once, twice, three times, or four times daily) or less frequently (e.g., once every other day, or once or twice a week). The dosage amounts described herein represent the individual amounts administered. When more than one compound is administered, the preferred dosage amounts correspond to the total amount of the compounds of the invention administered. The oral compositions described herein may contain about 10% to about 95% by weight of the active ingredient.

In general, suitable dosage ranges for intravenous (i.v.) administration of the individual components are 0.001 mg to 1000 mg per kilogram of body weight, 0.01 mg to 100 mg per kilogram of body weight, 0.1 mg to 50 mg per kilogram of body weight, and 1 mg to 10 mg per kilogram of body weight. In general, suitable dosage ranges for intranasal administration of the individual components are usually about 0.01 pg/kg to 1 mg/kg of body weight. In general, suppositories usually contain 0.01 mg to 50 mg of compound per kilogram of body weight and may contain active ingredient in the range of 0.5% to 10% by weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Such animal models and systems are well known in the art.

The invention also relates to pharmaceutical packs or kits comprising one or more containers filled with one or more compounds to be administered in carrying out the methods of the invention. Optionally associated with such containers may be a notice in a form specified by a government agency regulating the manufacture, use or sale of pharmaceutical or biological products, the notice reflecting approval by the agency of the manufacturer, use or sale for human administration. In certain embodiments, the kit contains more than one compound.

The compounds described herein are effective against susceptible or multidrug resistant bacteria, polymyxin resistant mutants, carbapenem resistant bacteria, methicillin resistant Staphylococcus aureus, vancomycin resistant Enterococci or multidrug resistant Neisseria gonorrhoeae.
The present invention is useful for treating infections caused by N. gonorrhoeae.

Examples of gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Candidatus liberibacter, Agrobacterium tumefaciens, Branhamella catarrhalis, Citrobacter diversus, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, and others. mirabilis, Salmonella typhimurium, Neisseria meningitidis, Serratia marcescens, Shigella sonnei, Shigella boydii, Neisseria gonorrhoeae, Acinetobacter baumannii, Salmonella enteritidis, Fusobacterium nucleatum nucleatum, Veillonella parvula, Actinobacillus actinomycetemcomitans, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Helicobacter pylori, Francisella tularensis, Yersinia pestis pestis, Vibrio cholerae, Morganella morganii, Edwardsiella tarda, Campylobacter jejuni, or Haemophilus influenza, Enterobacter cloacae, and many others. Other notable groups of gram-negative bacteria include cyanobacteria, spirochetes, green sulfur bacteria, and green non-sulfur bacteria.

Medically relevant gram-negative cocci include three organisms that cause sexually transmitted diseases (Neisseria gonorrhoeae), meningitis (Neisseria meningitidis), and respiratory symptoms (Moraxella catarrhalis).

There are numerous species of medically relevant gram-negative bacilli. Some of these are primarily responsible for respiratory disorders (Haemophilus influenza, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa), primarily urinary disorders (Escherichia coli, Enterobacter cloacae), and primarily gastrointestinal disorders (Helicobacter pylori, Salmonella enterica).

Gram-negative bacteria associated with hospital-acquired infections include Acinetobacter baumannii, which is responsible for bacteremia, secondary meningitis, and ventilator-associated pneumonia in intensive care units in hospital facilities. In one embodiment, the compounds and compositions of the present invention are effective against the following Gram-negative bacteria: E. coli, S. enterica, Klebsiella: K. pneumoniae, K. oxytoca; Enterobacter: E. cloacae, E. aerogenes; Acinetobacter: A. calcoaceticus, A. It is useful for treating infections with one or more of A. baumannii; Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Providencia stuartii; Proteus: P. mirabilis, P. vulgaris.

In one embodiment, the compounds of formula (I), (Ia) or (II) or pharma- ceutically acceptable salts thereof or compositions comprising the same are useful for the treatment of Pseudomonas infections, including P. aeruginosa infections, such as skin and soft tissue infections, gastrointestinal infections, urinary tract infections, pneumonia and sepsis.

In one embodiment, a compound of formula (I), (Ia) or (II) or a pharma- ceutically acceptable salt thereof or a composition comprising the same is useful for the treatment of Acinetobacter infections, including A. baumannii infections, such as pneumonia, urinary tract infections and sepsis.

In one embodiment, the compounds of formula (I), (Ia) or (II) or pharma- ceutically acceptable salts thereof or compositions comprising same are useful for the treatment of Klebsiella infections, including K. pneumoniae infections, e.g., pneumonia, urinary tract infections, meningitis and sepsis.

In one embodiment, the compounds of formula (I), (Ia) or (II) or pharma- ceutically acceptable salts thereof or compositions comprising the same are useful for the treatment of E. coli infections, including E. coli infections, such as bacteremia, cholecystitis, cholangitis, urinary tract infections, neonatal meningitis and pneumonia.

The compounds of the present invention may be produced by growing a strain of the microorganism Photorhabdus khanii DSM 3369 under controlled conditions. The compounds are obtained by fermentation and recovery in substantially pure form as described herein. In particular, the compounds may be produced by a strain of Photorhabdus P. khanii DSM 3369 during aerobic fermentation of a suitable culture medium under conditions hereinafter described. Media such as those used for the production of many antibacterial substances are suitable for use in this method for the production of the compounds.

One embodiment of the present invention comprises a method suitable for the production of antibiotic preparations, for example, of formula (II), by submerged aerobic fermentation of Photorhabdus khanii DSM 3369. The compound of formula (II) may be recovered from the fermentation broth by absorption on a resin and eluted from the resin by washing with solvents of different polarities. Purification may proceed by chromatographic separation techniques such as reversed-phase high performance chromatography (RP-HPLC).

Additional microorganisms capable of producing one or more compounds of the invention include mutant species that exhibit advantageous properties compared to species known in the art. Such bacterial strains can be generated by mutagenesis of a parent strain. Mutagenesis strategies and methods used to produce mutant strains of the invention, procedures for screening and isolating mutant bacterial strains, and media compositions are known in the art.

In a preferred embodiment, the cultivation of Photorhabdus khanii DSM 3369 for the production of the compound of formula (II) is carried out in a medium containing one or more absorbents along with readily assimilable carbon sources, nitrogen sources, inorganic salts and other organic components under suitable aeration conditions and with mixing in a sterile environment. The composition of the medium used in the production of the antibiotic formulation of the present invention is detailed in the Examples. (As used herein, the term "medium" refers to a mixture of synthetic or natural components. Generally, the medium contains a carbon source, a nitrogen source, trace elements such as inorganic salts, and, optionally, vitamins or other growth factors.)

Analogs of the compounds described herein may be produced biosynthetically using directly derived variants of the wild-type genomic sequence encoding the compounds. The genome sequence of Photorhabdus khanii DSM 3369 shows a match between the linear amino acid sequence of darobactin A, compound of formula (II) (WNWSKSF (SEQ ID NO: 1)), as shown in FIG. 4, and a portion of the genes belonging to an operon typically encoding RiPP (ribosomal translation system post-translationally modified peptide)-type antibacterial drugs. The operon includes a biosynthetic gene cluster encoding darA (encoding the precursor peptide of darobactin A, darobactin A, compound of formula (II)), darBCD (encoding apparently a transporter for the export of darobactin A), and darE (a radical SAM enzyme required for the generation of inactive C-C bonds, such as the tryptophan-lysine C-C bond of darobactin A), according to the nomenclature adopted by the inventors herein. The 7aa sequence of darobactin A is shown in the precursor peptide. Sequence alignment shows the presence of a dar operon in many Photorhabdus with a conserved core peptide.

We have obtained E. coli that are resistant to darobactin. These are located in BamA, an essential component of the Bam complex that helps fold and insert proteins into the outer membrane.

Ribosomal encoding of the darobactin A core peptide explicitly means that all amino acids are in the L-form. Ribosomal encoding allows the production of analogs of darobactin A by nucleotide substitution in the darA gene that encodes the precursor peptide. Such substitutions may be made using any of a variety of standard biochemical methods. Synthesis of oligonucleotides with both specific and random substitutions of nucleotides in the coding region of darobactin A yields large sequence fragments. These oligonucleotides are ligated with upstream and downstream sequences encoding the precursor peptide, cloned into an expression vector, and transformed into cells carrying the dar operon with the disrupted precursor peptide gene. Darobactin A analogs are isolated from clones of this recombinant library and tested for activity. Analogs with increased potency against bacteria or improved pharmacological properties may be isolated and developed into drugs.

Where technically appropriate, embodiments may be combined, and thus the present disclosure extends to all permutations/combinations of the embodiments provided herein. The examples herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Fermentation Photorhabdus khanii DSM3369 was inoculated into 10 ml of Luria Bertani broth (LBB) and incubated with shaking (200 rpm) in a 50 ml Falcon tube at 28° C. Ten milliliters of the overnight culture was inoculated into 1 L of Tryptic Soy Broth (TSB) in a 2 L Erlenmeyer flask and incubated at 28° C. with shaking (200 rpm) for 8-10 days.

Isolation Bacterial cells were removed by centrifugation (8,000×g, 10 min) and the supernatant was collected. The supernatant was mixed (4:1, vol/vol) with activated styrene-divinylbenzene resin (AMBERLITE XAD16N, SIGMA-ALDRICH) and then gently shaken overnight at ambient temperature. Unbound material was removed by decantation and the compound adsorbed on the resin, darobactin A (corresponding to formula (II)), was washed with DD water in an amount of 6 times the resin volume. Darobactin A was eluted with 4 times the resin volume of 50% methanol containing 0.1% (vol/vol) formic acid by gentle shaking on a rotary shaker for 30 min. The eluate was concentrated by rotary evaporation to remove the organic solvent. The remaining aqueous eluate was acidified with 0.1% (v/v) formic acid and then subjected to cation exchange (SP Sepharose XL, GE Healthcare) chromatography with a loose-resin bulk process. Activated cation exchange resin was added to the acidified aqueous eluate and gently shaken overnight at 4°C. Unbound material was decanted and the resin was washed with 0.1% (v/v) formic acid DD water. Antimicrobial activity was eluted with a step gradient of 50 mM ammonium acetate at pH 5, 50 mM ammonium acetate at pH 7, and 50 mM ammonium acetate at pH 8. Antimicrobial activity eluates were combined, lyophilized, and then resuspended in 0.1% (v/v) formic acid DD water. The resuspension was eluted with 4HiTrap HPLC column in series with elution with a step pH gradient.
A second cation exchange chromatography was performed on SP XL (5 mL per cartridge; total bed volume of 20 mL). Unbound material was washed with 0.1% (v/v) formic acid in DD water and then eluted with a step gradient of 50 mM ammonium acetate, pH 5, 50 mM ammonium acetate, pH 7, and 50 mM ammonium acetate, pH 8. The antibacterial active eluates were combined, lyophilized, and then resuspended in 0.1% (v/v) formic acid in DD water and subjected to reversed-phase high performance chromatography (RP-HPLC) on a C18 semi-preparative column (Agilent, C18, 5 μm; 250×10 mm, Restek) using a linear gradient starting from 2% B to 14% B with water + 0.1% (v/v) formic acid as solvent A/acetonitrile + 0.1% (v/v) formic acid as solvent B at a flow rate of 5 mL/min for 14 min, and UV detection from 210 to 400 nm monitored by a diode array detector to give Darobactin A at 12.25 min (purity: 95% UV).

Structure of Darobactin A Darobactin A was isolated as an amorphous white powder after lyophilization.

Chemical tests for Darobactin A showed a positive reaction with ninhydrin reagent, indicating the presence of a primary amine. It also showed a positive reaction with the ferric chloride test, indicating the presence of phenol.

Direct injection of 1 μg/ml of Darobactin A resulted in high resolution electrospray ionization mass spectrometry (HRE) data obtained on a THERMO SCIENTIFIC LTQ Orbitrap XL hybrid ion trap mass spectrometer.
SIMS) showed a [M+2H] ²⁺ peak at m/z 483.70874 and a [M ₊ H] ⁺ peak at m/z _966.41046 . The isotopic pattern showed no Cl or Br present. The exact mass and _{isotopic distribution were consistent with the elemental formula C47H55O12N11 .}

One- and two-dimensional magnetic resonance (1D and 2D NMR) analysis on a Bruker 500 MHz of 5 mg of Darobactin A dissolved in 500 μL of water + 3% (v/v) deuterium oxide and 0.25% (v/v) deuterated formic acid showed a peptide and revealed the presence of a phenylalanine, two serines, a lysine, two tryptophans and an asparagine. COSY, TOCSY, NOESY and HMBC spectra allowed the linkage of the different amino acids by peptide bonds to be determined and revealed an unusual lysine-tryptophan bridge. This bond appears to arise by a post-translational modification previously described by R. Schramma et al. (2015), involving a covalent bond between two inactive carbons in the side chains of lysine and tryptophan.

Acid hydrolysis of darobactin A followed by derivatization with Murphy's reagent and LC-MS amino acid determination experiments and fragmentation pattern analysis of the whole compound by tandem mass spectrometry (MS/MS) confirmed the peptide pattern indicative of a novel natural product.

Antibacterial Spectrum The minimum inhibitory concentration (MIC) of Darobactin A was determined by broth microdilution assay. The following test organisms were used to evaluate the spectrum of Darobactin A: Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella Typhimurium, Shigella sonnei, Enterobacter cloacae, and Escherichia coli. cloacae), Bacteroides fragilis, Lactobacillus reuteri, and Enterococcus faecalis.

Test organisms were inoculated into 5 mL of Mueller-Hinton II broth (MHIIB) for aerobic conditions or Brain Heart Infusion broth (BHI) for anaerobic conditions and grown until a cell density of OD ₆₀₀ =0.1-0.9 was reached. The cell suspension was diluted to OD ₆₀₀ =0.001 and 96 μL of bacterial culture was added to 4 μL of Darobactin A (final concentration 0.5-128 μg/mL) in a 96-well plate. After 20 h of incubation at 37° C., growth of the test organisms was determined visually (Table 1).

Cytotoxicity HepG2 and FaDu cells were cultured in 20 mL of Minimum Essential Medium (MEM) + 10% fetal bovine serum (FBS) and 1% antibacterial and antifungal antibiotics in a 75 ^cm2 tissue culture flask and incubated at 37°C, 5% CO2 for 5 days (until the cells reached 70% confluence). The growth medium was removed and the cells were washed with 5 mL of 1x phosphate buffered saline (PBS). After washing, the PBS was removed and ₂ mL of 1x EDTA solution containing 0.25% trypsin was added. The cells were incubated for 2 minutes at 37°C, 5% _CO2 to detach the cells from the flask. The trypsin was inactivated by the addition of 6 mL of culture medium to the flask and the cells were detached by gentle pipetting to avoid clumping. Cells were counted and diluted to ^2x105 cells/mL in MEM + 10% FBS, and 100 μl of this suspension was added to a clear flat-bottom tissue culture treated 96-well plate. The plate was incubated overnight at 37°C, 5% _CO2 to allow the cells to adhere to the plate.

In separate clear round-bottom non-tissue culture treated 96-well plates, 4 μl of Darobactin A was mixed with 96 μl of culture medium. Supernatants were removed from each tissue culture plate and culture medium containing Darobactin A was transferred from the round-bottom plate to the tissue culture plate. Cells were incubated in the presence of Darobactin A for 3 days at 37° C., 5% _CO2 , at which point 5 μl of 3 mM resazurin (Alamar Blue) was added. Plates were incubated for 3 hours at 37° C., 5% _CO2 . Viability of each cell line was assessed by reading fluorescence (554 nm/590 nm) on a plate reader (Table 2).

Killing activity of Darobactin A E. coli was inoculated into 5 mL of Mueller-Hinton II medium (MHIIB) and grown to OD ₆₀₀ =0.1-0.9. The cell suspension was diluted to OD ₆₀₀ =0.001. 98 μL of E. coli culture was treated with 2 μL of Darobactin A (16 μg/mL, equivalent to 2×MIC) in a 96-well plate and incubated at 37° C. for 20 h. E. coli culture was plated on Luria Broth agar without antibiotics and colony forming units (CFU) were counted for viable cell count ( FIG. 3 ).

Antagonism assay by lipopolysaccharide (LPS) Darobactin A is a large compound with a molecular weight of 966 Da. The entry limit for Gram-negative bacteria is approximately 500 Da. Therefore, it was thought that Darobactin A might act on the cell surface similarly to polymyxins that target lipopolysaccharide (LPS). To test this, the activity of Darobactin A was tested in the presence of LPS. E. coli was inoculated into 5 mL of MHIIB and grown until the cell density reached OD ₆₀₀ =0.1-0.9. The cell suspension was diluted to OD ₆₀₀ =0.002 in 2×MHIIB. 40 μL of LPS solution (final concentration range 0-800 μg/mL) and 2 μL of 2×MIC antimicrobial solution (final concentrations: Polymyxin; 0.5 μg/mL, Ampicillin; 16 μg/mL, Darobactin A; 16 μg/mL) were mixed in a 96-well plate and filled up to 50 μL with sterile DI water. The LPS and antimicrobial solutions were mixed with 50 μL of E. coli cell suspension and incubated at 37° C. for 20 hours. E. coli growth was determined visually (Table 3).

Animal Efficacy Darobactin A was tested in two animal efficacy models (Figure 5). In the sepsis model, mice were injected (intraperitoneally) with ¹⁰⁶ bacteria in 5% mucin as inoculum causing 100% mortality in the untreated group within 24 hours (Figures 5A, 5C). One hour after infection, mice were treated with gentamicin 20 mg/kg as a positive control or with various dose levels of Darobactin A in three groups. Survival was monitored over a period of five days. Darobactin A effectively treated the infection, showing 100% survival at 2.5 mg/kg against both the commonly used ATCC25922 strain and the clinical isolate AR350 resistant to polymyxins (Figure 5A). After 5 hours, tail blood was collected for CFU determination, showing a significant reduction in Darobactin A-treated E. coli (Figure 5B). At 1 mg/kg, Darobactin A still extended survival time, but was no longer 100% cured (Figure 5A). Darobactin A was tested at 50 mg/kg against P. aeruginosa sepsis using a laboratory strain resistant to polymyxin, showing 100% survival (Figure 5C). Darobactin A was then tested for efficacy in a neutropenic mouse thigh model. Mice were given cyclophosphamide to induce neutropenia (day -3, day 0), then infected with ¹⁰⁶ CFU E. coli AR350 in the right thigh, with treatment starting 2 hours after infection. Untreated controls, 4 mice per group, were bagged at 0, 2, and 24 hours to monitor the progression of the infection. In four mice per treatment group, a single 50 mg/kg injection of Darobactin A (ip) was compared with three doses of 25 mg/kg Darobactin A (ip) given at 2, 8, and 14 hours after infection, and a single 20 mg/kg injection of Gentamicin (ip) at 2 hours. Mice were bagged at 24 hours and CFUs were counted by homogenizing aseptically removed thighs and plating them on agar plates (Figure 5D). Darobactin A at 50 mg/kg reduced the infection burden 10-fold compared to untreated controls, and three doses of 25 mg/kg reduced infection 100-fold, including below the limit of detection in two mice.

Thus, Darobactin A was able to cure mice with E. coli sepsis at 2.5 mg/kg (ip), ATCC25922 and polymyxin-resistant clinical isolate AR350, n=3/treatment group (treated 1 h after infection). At 1 mg/kg (ip), Darobactin A extended survival time and significantly reduced blood CFU burden at 5 h (A, B).

The terms and expressions used in this specification are used as descriptive terms and expressions and not as limiting terms, and there is no intention in the use of such terms and expressions to exclude any equivalents or portions of the features shown and described. In addition, it will be apparent to those skilled in the art that, given the specific embodiments of the invention described, other embodiments incorporating the concepts disclosed herein may be used without departing from the spirit and scope of the invention. The described embodiments are therefore to be considered in all respects as illustrative only and not limiting.

Claims (21)

Formula (II):

and/or a pharma- ceutically acceptable salt thereof. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to claim 1 or a pharma- ceutical acceptable salt thereof. The pharmaceutical composition of claim 2, further comprising at least one pharma- tically acceptable carrier, excipient, or diluent. The pharmaceutical composition according to claim 2 or 3, which is administered locally, systemically, parenterally, subcutaneously, transdermally, rectally, orally, intravaginally, intranasally, intrabronchially, intraocularly, intraaurally, intravenously, intramuscularly, or intraperitoneally. The pharmaceutical composition according to any one of claims 2 to 4, further comprising at least one additional therapeutic agent. A method for producing the pharmaceutical composition according to any one of claims 2 to 5, comprising the steps of:
The compound of formula (II) or a pharma- ceutically acceptable salt thereof can be obtained by culturing a microorganism capable of producing the compound in a culture medium.
Production method. The method according to claim 6, wherein the microorganism is Photorhabdus khanii DSM 3369. The pharmaceutical composition according to any one of claims 2 to 5 for treating an infectious disease in an animal caused by a gram-negative bacterium. Use of a compound according to claim 1 or a pharma- ceutical acceptable salt thereof in the manufacture of a medicament for the treatment, amelioration or prevention of a bacterial infection or disease in a subject. The use according to claim 9, wherein the bacteria causing the bacterial infection comprise gram-negative bacteria. The gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa, Candidatus liberibacter, Agrobacterium tumefaciens, Branhamella catarrhalis, Citrobacter diversus, Enterobacter aerogenes, Klebsiella pneumoniae, Proteus mirabilis, and the like. mirabilis, Salmonella typhimurium, Neisseria meningitidis, Serratia marcescens, Shigella sonnei, Shigella boydii, Neisseria gonorrhoeae, Acinetobacter baumannii, Salmonella enteritidis, Fusobacterium nucleatum nucleatum, Veillonella parvula, Actinobacillus actinomycetemcomitans, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Helicobacter pylori, Francisella tularensis, Yersinia pestis pestis, Vibrio cholerae, Morganella morganii, Edwardsiella tarda, Campylobacter jejuni, Haemophilus influenza, or Enterobacter cloacae. The use according to claim 10, wherein the bacterium is selected from the group consisting of: The use according to any one of claims 9 to 11, wherein the bacteria causing the bacterial infection comprise multi-drug resistant bacteria. The use according to any one of claims 9 to 12 , wherein the bacteria causing the bacterial infection comprise polymyxin resistant mutant bacteria. 14. The use according to any one of claims 9 to 13, wherein the bacteria causing the bacterial infection comprise carbapenem-resistant bacteria, methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci or multidrug-resistant Neisseria gonorrhoeae. The use according to any one of claims 9 to 14, wherein the bacterial infection is a respiratory infection, a skin or skin structure infection, a urinary tract infection, an intraperitoneal infection, a blood infection or a gastrointestinal infection. The use according to claim 9, wherein the disease is selected from the group consisting of inflammatory skin diseases, inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, and celiac disease. The use according to any one of claims 9 to 15, wherein the subject is a mammal. The use according to claim 16, wherein the subject is a human. The use according to claim 16, wherein the subject is a non-human. The use according to any one of claims 9 to 18, wherein the bacteria causing the bacterial infection comprise bacteria that are sensitive to antibacterial drugs or multi-drug resistant bacteria. The use according to any one of claims 9 to 19, wherein the pharmaceutical agent is administered by a step including local administration, systemic administration, parenteral administration, subcutaneous administration, transdermal administration, rectal administration, oral administration, intravaginal administration, intranasal administration, intrabronchial administration, intraocular administration, intraaural administration, intravenous administration, intramuscular administration, or intraperitoneal administration.

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